Search

Aim of the study was to evaluate the late-pregnancy and perinatal outcomes of patients with threatened miscarriage in the first trimester.An observational cohort study was performed on 81 pregnant women. Subjects were divided into two groups: 1) no bleeding; 2) threatened miscarriage. Patients were followed up until delivery and each materno-fetal complication was registered.Threatened miscarriage was associated with increased risk of preterm delivery, placenta previa, pregnancy induced hypertension/preeclampsia (PE), low birth weight (LBW) and neonatal intensive care unit (NICU) admission. There were no significantly differences between the 2 groups with regard to preterm prelabour rupture of membranes (PPROM), CESAREAN section, retained placenta, perinatal death and intrauterine growth restriction (IUGR). About immediate neonatal outcomes, mean birth weights were lower (≈ 200 g) in the study group (group 2), while no significant difference in the APGAR score between the two groups was noted.Our study suggests that threatened miscarriage in the first trimester is correlated with an increased incidence of late-pregnancy and perinatal complications and, therefore, these pregnancies should be considered as high risk ones.

Transplantation of hematopoietic cells from a healthy individual (allogeneic hematopoietic cell transplantation (allo-HCT)) demonstrates that adoptive immunotherapy can cure blood cancers: still, post-transplantation relapses remain frequent. To explain their drivers, we analyzed the genomic and gene expression profiles of acute myeloid leukemia (AML) blasts purified from patients at serial time-points during their disease history. We identified a transcriptional signature specific for post-transplantation relapses and highly enriched in immune-related processes, including T cell costimulation and antigen presentation. In two independent patient cohorts we confirmed the deregulation of multiple costimulatory ligands on AML blasts at post-transplantation relapse (PD-L1, B7-H3, CD80, PVRL2), mirrored by concomitant changes in circulating donor T cells. Likewise, we documented the frequent loss of surface expression of HLA-DR, -DQ and -DP on leukemia cells, due to downregulation of the HLA class II regulator CIITA. We show that loss of HLA class II expression and upregulation of inhibitory checkpoint molecules represent alternative modalities to abolish AML recognition from donor-derived T cells, and can be counteracted by interferon-gamma or checkpoint blockade, respectively. Our results demonstrate that the deregulation of pathways involved in T cell-mediated allorecognition is a distinctive feature and driver of AML relapses after allo-HCT, which can be rapidly translated into personalized therapies.

T cell receptor (TCR)–based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms’ tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01–restricted TCRs, three that were specific to the less explored immunodominant WT1 37–45 and two that were specific to the noncanonical WT1 −78–64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant ( TRAC ) locus with TCR β constant ( TRBC ) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT1 37–45 -specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

Adaptation is the ability of cells, tissues and organisms to rapidly and reversibly modify their properties to maximize fitness in a changing environment. The activity of immune-system components unfolds in the remarkably heterogeneous milieus to which they are exposed in different tissues, during homeostasis or during various acute or chronic pathological states. Therefore, adaptation is essential for immune cells to tune their responses to a large variety of contexts and conditions. The adaptation of immune cells reflects the integration of multiple inputs acting simultaneously or in a temporal sequence, which eventually leads to transcriptional reprogramming and to various functional consequences, some of which extend beyond the duration of the stimulus. A range of adaptive responses have been observed in both adaptive immune cells and innate immune cells; these are referred to with terms such as ‘plasticity’, ‘priming’, ‘training’, ‘exhaustion’ and ‘tolerance’, among others, all of which can be useful for defining a certain immunological process or outcome but whose underlying molecular frameworks are often incompletely understood. Here we review and analyze mechanisms of adaptation and memory in immunity with the aim of providing basic concepts that rationalize the properties and molecular bases of these essential processes.

Tight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8+ T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8+ T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors., Acquisition of dying tumor cell-associated antigens is an essential step for the initiation of anti-tumor immune response by conventional type 1 dendritic cells (cDC1). Here the authors show that the loss of TIM4 expression in lung tumor associated cDC1 is associated with less efficient uptake of cell associated antigens and reduction of CD8 + T cell activation in advanced lung tumors.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing., An immune suppressive tumor microenvironment (TME) is a limitation for immunotherapy. Here the authors show that, in a B cell acute lymphoblastic leukemia mouse model, gene-based delivery of IFNα reprograms the leukemia-induced immunosuppressive TME into immunostimulatory and enhances T-cell responses.

The ST18 gene was proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis was missing so far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 mRNA and protein were detectable neither in the normal liver nor in cultured hepatoblasts, but were readily expressed following subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as co-culture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors, caused rapid tumor involution, associated with pervasive morphological changes, proliferative arrest and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in the hepatoblasts. Finally, RNA-seq profiling of ST18-depleted tumors prior to involution revealed down-regulation of inflammatory response genes, pointing to the suppression of NF-kB-dependent transcription.CONCLUSION: ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. This article is protected by copyright. All rights reserved.

Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. We analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of draining lymph nodes, where they extensively interact with CD11b+Ly6Chi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon– and CCR2-dependent fashion, and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment, or impairment of their nitric oxide–producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. Our results identify inflammatory monocytes as critical gatekeepers that restrain antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.

In addition to performing well-defined effector functions, neutrophils are now recognized as versatile and sophisticated cells with critical immunoregulatory roles. These include the release of a variety of proinflammatory or immunosuppressive cytokines, as well as the expression of genes with regulatory functions. Neutrophils share broad transcriptional features with monocytes, in keeping with the close developmental relation between the two cell types. However, neutrophil-specific gene expression patterns conferring cell type-specific responses to bacterial, viral or fungal components have been identified. Accumulating evidence suggest that these differences reflect the peculiar epigenomic and regulatory landscapes of neutrophils and monocytes, in turn controlled by the specific lineage-determining transcription factors shaping their identity. In this review, we will describe current knowledge on how neutrophil identity and function are controlled at the molecular level, focusing on transcriptional and chromatin regulation of neutrophil development and activation in response to inflammatory stimuli.

Inflammation is a multistep process triggered when innate immune cells - for example, DCs - sense a pathogen or injured cell or tissue. Edema formation is one of the first steps in the inflammatory response; it is fundamental for the local accumulation of inflammatory mediators. Injection of LPS into the skin provides a model for studying the mechanisms of inflammation and edema formation. While it is known that innate immune recognition of LPS leads to activation of numerous transcriptional activators, including nuclear factor of activated T cells (NFAT) isoforms, the molecular pathways that lead to edema formation have not been determined. As PGE2 regulates many proinflammatory processes, including swelling and pain, and it is induced by LPS, we hypothesized that PGE2 mediates the local generation of edema following LPS exposure. Here, we show that tissue-resident DCs are the main source of PGE2 and the main controllers of tissue edema formation in a mouse model of LPS-induced inflammation. LPS exposure induced expression of microsomal PGE synthase-1 (mPGES-1), a key enzyme in PGE2 biosynthesis. mPGES-1 activation, PGE2 production, and edema formation required CD14 (a component of the LPS receptor) and NFAT. Therefore, tissue edema formation induced by LPS is DC and CD14/NFAT dependent. Moreover, DCs can regulate free antigen arrival at the draining lymph nodes by controlling edema formation and interstitial fluid pressure in the presence of LPS. We therefore suggest that the CD14/NFAT/mPGES-1 pathway represents a possible target for antiinflammatory therapies.

Summary Histone methyltransferases catalyze site-specific deposition of methyl groups, enabling recruitment of transcriptional regulators. In mammals, trimethylation of lysine 4 in histone H3, a modification localized at the transcription start sites of active genes, is catalyzed by six enzymes (SET1a and SET1b, MLL1–MLL4) whose specific functions are largely unknown. By using a genomic approach, we found that in macrophages, MLL4 (also known as Wbp7) was required for the expression of Pigp, an essential component of the GPI-GlcNAc transferase, the enzyme catalyzing the first step of glycosylphosphatidylinositol (GPI) anchor synthesis. Impaired Pigp expression in Wbp7 −/− macrophages abolished GPI anchor-dependent loading of proteins on the cell membrane. Consistently, loss of GPI-anchored CD14, the coreceptor for lipopolysaccharide (LPS) and other bacterial molecules, markedly attenuated LPS-triggered intracellular signals and gene expression changes. These data link a histone-modifying enzyme to a biosynthetic pathway and indicate a specialized biological role for Wbp7 in macrophage function and antimicrobial response.

SummaryThe transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.

The key driving force underlying cell identity is represented by the complex and dynamic interplay between cell-intrinsic, lineage-restricted developmental pathways on the one hand, and cell-extrinsic, tissue-specific microenvironmental signals on the other. In this context, macrophages are a paradigmatic cell population whose functional specialization in vivo reflects the impact of the local microenvironment on the intrinsic differentiation program, leading to a variety of specialized macrophage types in different tissues and conditions; however, how this is translated into a biological outcome is not appreciably understood. The kind of investigations described in this Viewpoint aim to explore the inner determinants of cell identity and functional diversification at a genomic level; mechanisms that permit plastic cell types, like macrophages, to adapt to different environments. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPβ recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.

Cellular differentiation progresses through an ordered cascade of events involving cell autonomous and micro-environment regulated expression or activation of transcription factors (TFs). Lineage-determining and stimulus-activated TFs collaborate in specifying the transcriptional programs of differentiating cells through the establishment of appropriate genomic repertoires of active or poised cis-regulatory elements, which can eventually be altered by environmental changes to generate transient or persistent functional states. Here, we rationalize available genomic and functional data into a mechanistic model whereby terminal differentiation proceeds first through the establishment of a regulatory landscape that is broadly shared among all cell types within a given lineage; and then through the selective activation of a more restricted set of regulatory elements that specify the unique transcriptional outputs of individual cell types. In this scheme, the interplay between cell-autonomous and microenvironment-regulated TFs is highly complex, with several documented variants.

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modification status at their IL-10 genomic locus. We analyzed posttranslational modifications of histones associated with genes that are active, repressed, or poised for transcriptional activation, including H3K4me3, H4Ac, H3K27Ac, and H3K4me1 marks. Differently from autologous IL-10–producing monocytes, none of the marks under evaluation was detected at the IL-10 locus of resting or activated neutrophils from healthy subjects or melanoma patients. By contrast, increased H3K4me3, H4Ac, H3K4me1, and H3K27Ac levels were detected at syntenic regions of the IL-10 locus in mouse neutrophils. Altogether, data demonstrate that human neutrophils, differently from either monocytes or mouse neutrophils, cannot switch on the IL-10 gene because its locus is in an inactive state, likely reflecting a neutrophil-specific developmental outcome. Implicitly, data also definitively settle a currently unsolved issue on the capacity of human neutrophils to produce IL-10.

SummaryAccording to current models, once the cell has reached terminal differentiation, the enhancer repertoire is completely established and maintained by cooperatively acting lineage-specific transcription factors (TFs). TFs activated by extracellular stimuli operate within this predetermined repertoire, landing close to where master regulators are constitutively bound. Here, we describe latent enhancers, defined as regions of the genome that in terminally differentiated cells are unbound by TFs and lack the histone marks characteristic of enhancers but acquire these features in response to stimulation. Macrophage stimulation caused sequential binding of stimulus-activated and lineage-determining TFs to these regions, enabling deposition of enhancer marks. Once unveiled, many of these enhancers did not return to a latent state when stimulation ceased; instead, they persisted and mediated a faster and stronger response upon restimulation. We suggest that stimulus-specific expansion of the cis-regulatory repertoire provides an epigenomic memory of the exposure to environmental agents.

The lipopolysaccharide is the major component of Gram-negative bacteria outer membrane. LPS comprises three covalently linked regions: the lipid A, the rough core oligosaccharide, and the O-antigenic side chain determining serotype specificity. Wild-type LPS (sLPS) contains the O-antigenic side chain and is referred to as smooth. Rough LPS (rLPS) does not contain the O-side chain. Most wt bacteria and especially wt Enterobacteriaceae express prevalently the sLPS form although some truncated rLPS molecules always reach the external membrane. The two sLPS and rLPS forms are used almost indistinctly to study the effects on innate immune cells. Nevertheless, there is evidence that their mechanism of action may be different. For instance, while sLPS requires CD14 for the initiation of both MyD88-dependent and independent signal transduction pathways at least at low doses, rLPS leads to MyD88-dependent responses in the absence of CD14 even at low doses. Here we have identified additional differences in the signaling capacity of the two LPS species in the mouse. We have found that rLPS, diversely from sLPS, is capable of activating in dendritic cells (DCs) the Ca 2+/calcineurin and NFAT pathway in a CD14-independent manner, moreover it is also capable per se of activating the inflammasome and eliciting IL-1β secretion independent of the presence of additional stimuli required instead for sLPS. The ability of rLPS of activating the inflammasome in vitro has as a direct consequence a higher efficiency of rLPS-exposed DCs in activating natural killer (NK) cells compared to sLPS-exposed DCs. However, diversely from possible predictions, we found that the different efficiencies of the two LPS species in eliciting innate responses are almost nullified in vivo. Therefore, sLPS and rLPS induce nearly similar in vivo innate responses but with different mechanisms of signaling. © 2011 Elsevier B.V.

Innate and adaptive defense mechanisms participate to immunological protection. Innate immune cells like macrophages, neutrophils and dendritic cells (DCs) regulate the first part of the response, which is rapidly activated at the site of infection. At this stage, phagocytic cells eliminate pathogens from the infected tissue and additionally establish a local inflammatory state by releasing proinflammatory cytokines (e.g. TNF-alpha, IL-1beta) and lipid mediators (PGE2). Subsequently, activated tissue-resident DCs acquire a migratory phenotype and reach the draining lymph node to prime antigen-specific T cells for the initiation of adaptive immunity. Innate immune cells detect invading pathogens through a set of receptors recognizing conserved patterns that are unique to microbes. Specifically, Gram-negative bacteria are perceived by means of TLR4 and CD14, which act as a receptor complex for lipopolysaccharide (LPS), the main constituent of the bacterial cell wall. Upon LPS recognition, TLR4 and CD14 cooperate to trigger signal transduction cascades leading to activation of the transcription factors NF-kappaB, AP-1 and IRF3, which in turn promote expression of proinflammatory genes. Despite the signaling pathways induced by LPS have been mainly described in macrophages, it is widely believed that they do not differ significantly across innate immune cell types, including DCs. However, DCs do show very specific biological responses to LPS stimulation such as the ability to release IL-2, a key factor for Natural Killer (NK) cell activation. We found that CD14 triggers a pathway involving Ca++ influx, Src family kinases (SFKs) and PLC-gamma2 that leads to NFAT activation in LPS-stimulated DCs. Notably, activation of this pathway was DC-specific and TLR4-independent. We also showed that CD14-NFAT pathway promotes the expression of pro-apoptotic genes that induce apoptosis of activated DCs as a strategy to prevent excessive immune responses. Our work revealed several novel aspects as: (i) the unexpected capability of CD14 to function independently of TLR4 as a transducing receptor; (ii) the previously unrecognized biological role of NFAT in DCs; (iii) a molecular explanation for the differential life cycle of DCs and macrophages after activation, consistent with their specific physiological role in innate immunity. Additional work revealed that LPS-induced NFAT activation in DCs is also necessary for the efficient synthesis of PGE2, a crucial lipid mediator regulating many proinflammatory processes such as swelling and pain. We also showed that tissue edema formation induced by subcutaneous administration of LPS is CD14-NFAT-dependent, and that DCs play a major role in this process. Not only these results highlight another crucial biological function of this signal transduction pathway in DCs, but they also point to a key direct role of this cell type in the proinflammatory response to LPS. These data suggest that CD14 signaling in DCs may constitute a valuable target for the development of a novel class of anti-inflammatory compounds devoid of the secondary effects observed with COX-2 inhibitors.